Project Summary Recent advancements in high-throughput sequencing techniques have provided many insights into one of the most fundamental questions in modern biology: gene regulation. Such ?seq? techniques have elucidated enhancer distributions in the genome, revealed distinct chromosome conformation in a nucleus, and identified hotspots within the genome that are highly associated with disease phenotypes. And yet, there remain unanswered questions. For example, while spatial boundary of gene expression pattern has been studied extensively, the kinetics of gene expression is not as well understood. We know that enhancer and promoter need to interact with each other to produce mRNA, but it is not clear how often, or how long they need to interact to initiate transcription. Moreover, while complete removal of an enhancer is known to abolish gene expression, the effect of moderate disruptions in enhancer or enhancer-promoter interactions on development is yet to be characterized. I believe that recent advancement in imaging techniques can provide equally exciting discoveries in gene regulation by complementing the ?seq? techniques. In the next five years, I envision achieving molecular-level understanding of dynamic gene control that ensures normal development, using combination of live imaging, quantitative image analysis, and mathematical modeling. Using early Drosophila embryo, I propose to study three aspects of gene control that affect developmental phenotypes: (1) multiple enhancers contributing to developmental robustness by reducing cell-to-cell variability in transcription, (2) characteristics of enhancer-promoter interactions that guarantee transcriptional initiation, and (3) modulations in chromosomal loop domain affecting the error-rate of enhancer-promoter interactions and leading to variable developmental phenotypes. The proposed study will provide exciting new perspectives on the field of gene regulation during development. The insights obtained from this interdisciplinary study will be relevant to all other gene expression phenomena underlying metabolism, disease, and other aspects of human physiology.